Composition comprising zoanthus sp. extract with anti-fouling activity and a method thereof

ABSTRACT

The present invention relates to a composition useful as an anti-fouling agent by inhibiting microbial growth, said composition comprising extract and/or its fractions from Zoanthus sp. and optionally de-odorizing agents and a method of producing said extract and its fractions having about 100% anti-bacterial activity fouling microbes.

FIELD OF THE PRESENT INVENTION

[0001] The present invention relates to a composition useful as ananti-fouling agent by inhibiting microbial growth, said compositioncomprising extract and/or its fractions from Zoanthus sp. and optionallyde-odorizing agents and a method of producing said extract and itsfractions having about 100% anti-bacterial activity fouling microbes.

BACKGROUND AND PRIOR ART REFERENCES

[0002] Chemical antifouling defense strategies of marine organisms havebeen studied either directly against the target fouling organisms suchas bacteria, fungi, algae, invertebrate larvae, etc. (Amade et ai.,1987,Mar.Biol.94, 271-275; Slattery et al.,1995, J. Exp. Mar. Biol. Ecol,190, 61-77; Shellenberger and Ross, 1998, Northwest Science, 72(1),23-33) or using other fouling measures like settlement inhibition,larval moulting rates, photosynthesis, etc. (Kon-ya et al., 1994,Fish.Sci, 60(6), 773-775; Kato et al., 1995, Tetrahedron letters,36(12), 2133-2136; Mizobuchi et al., 1996, Fisheries Sci, 62(1), 100;Becerro et al., 1997, J. Chem. Ecol, 23(6), 1527-1547).

[0003] Information on secondary metabolite production m marine organsdeals mostly with isolation and characterisation of the compounds andvery little is known about their ecological and biological importance.In recent years, some attempts have been made in this direction on a fewgroups such as soft corals, gorgonians, algae, sponges, bryozoans, etc.(Targett, 1988, in Marine Biodeterioration, M. F. Thompson., R.Sarojini., R. Nagabhushanam., A A Balkema eds, Rotterdam, 607-617;Standing et al., 1984, J. Chem. Ecol, 10, 823-824; Davis et al., 1991,Mar. Ecol, Prog. Ser, 72, 117-123; Vitalina et al., 1991, in Bioactivecompounds from marine organisms, M. F. Thompson., R. Sarojini., RNagabhushanam eds, Oxford and IBH Publishing Co. Pvt. Ltd, 63-68;Szewzyk et al., 1991, Mar. Ecol. Prog. Ser, 75, 259-265) leaving manyother groups unstudied. Zoanthid (Phylum Coelenterata; FamilyZoanthidae) is one such group which has received very little attentionwith respect to chemical defense strategies. Zoanthids are benthic,sessile, colony forming organisms, comprising of thousands of individualpolyps. Several chemical compounds have been isolated from zoanthids,possessing various pharmacological properties (Moore and Scheuer, 1971,Science, 172, 495-498; Cimino et al., 1973,; Sturaro et al., 1982; Raoet al., 1985, J.Org.Chem, 50, 3757-3760; Bakus et al., 1986,J.Chem.Ecol, 12, 951-987; Quinn, 1988, in Bioorganic Marine Chemistry,P. J. Scheuer ed. Vol 2, 25-27; Shigemori et al., 1999, J. Nat. Prods,62(2), 373). However, very little is known about the functions of thesemetabolites in their natural habitat. In the Anjuna beach of Goa (westcoast of India), they occur as dense mats and colonise a largeintertidal area. The main focus of the present Invention{circumflex over( )} therefore, is to ujidjrjtjadj{circumflex over ( )}e{circumflex over( )}h{circumflex over ( )}nucal antifouling defense properties againstcommon micro-fouling organisms viz. bacteria and diatoms utilising theextract of Zoanthus sp and in turn for identifying a\naturaJ productuseful in antifouling technqlojgy.

[0004] Any surface exposed to marine environment undergoes sequentialchanges that begins with the adsorption of a layer of bio-polymersfollowed by the formation of a bacterial film, growth of pinnate diatomsand protozoans (Baier, 1984, in Marine Biodeterioration: AnInterdisciplinary Study, J. D. Costlow and R. C. Tipper, ed, NavalInstitute Press, Anapolis, Md., 57-62; Mitchell and Kirchman, 1984, inMarine Biodeterioration: An Interdisciplinary Study, J. D. Costlow andR. C. Tipper, ed, Naval Institute Press, Anapolis, Md., 49-56 ; Wahl,1989, Mar. Ecol. Prog. Ser, 58, 175-189). Consequently, the variety ofbacterial species forming the microbial film may influence, inhibit orat times have no effect upon the later settling species which constitutethe invertebrate larvae that settle and metamorphose into adults (Bakuset al., 1986, J. Chem.Ecol, 12, 951-987). In most cases where it hasbeen shown that a microbial film is necessary for settling of larvae andalgal propagules, inhibition of bacterial growth would prevent themicrobial film formation and in turn inhibit larval settlement,ultimately leaving the surfaces free of epibionts.

[0005] In this effort, the test animals were collected and the crudemethanol extract was prepared. The antifouling potential of the extractwas then explored against known fouling diatoms and bacteria in thelaboratory. After testing the activity of the crude extract, it wasfractionated using solvents of increasing polarity. These fractions weretested against the fouling diatoms. The results of the evaluationreveals that the extract of Zoanthus sp has the antifouling property.

OBJECTS OF THE PRESENT INVENTION

[0006] The main object of the present invention is to develop acomposition comprising an extract from Zoanthus species.

[0007] Another main object of the present invention is to develop acomposition having anti-fouling property.

[0008] Yet another object of the present invention is to developfractions from extract of animal Zoanthus species.

[0009] Still another object of the present invention is to fractionatethe crude extract using solvents of increasing polarity, viz; petroleumether, chloroform, n-butanol and the aqueous fraction.

[0010] Still another object of the present invention is to identify thefraction which is most effective against fouling diatoms.

[0011] Still another object of the present invention is to develop amethod of preparing composition comprising an extract from Zoanthusspecies.

[0012] Still another object of the present invention is to develop amethod of preparing said composition having 100% anti-bacterialactivity.

[0013] Still another object of the present invention is to develop amethod of preparing said composition having 100% microbial growthinhibitory activity against fouling microbes.

[0014] Still another object of the present invention is to develop amethod of preparing said composition with anti-microbial activitywherein, said activity is particularly effective in marine environment.

[0015] Still another object of the present invention is to develop amethod of preparing said composition with anti-microbial activitywherein, said activity is particularly effective against bacteria anddiatoms.

SUMMARY OF THE PRESENT INVENTION

[0016] The present invention relates to a composition useful as ananti-fouling agent by inhibiting microbial growth, said compositioncomprising extract and/or its fractions from Zoanthus sp. and optionallyde-odorizing agents and a method of producing said extract and itsfractions having about 100% anti-bacterial activity fouling microbes.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0017] Accordingly, the present invention relates to a compositionuseful as an anti-fouling agent by inhibiting microbial growth, saidcomposition comprising extract and/or its fractions from Zoanthus sp.and optionally de-odorizing agents and a method of producing saidextract and its fractions having about 100% anti-bacterial activityfouling microbes.

[0018] In one embodiment of the present invention, a compositioncomprising extract and/or its fractions from Zoanthus sp, and optionallyde-odorizing agents wherein said composition is useful as ananti-fouling agent against microbes.

[0019] In another embodiment of the present invention, wherein saidextract and its fractions are prepared by using either polar ornon-polar solvent.

[0020] In yet another embodiment of the present invention, whereinanimal species preferably used for said activity is CoelenterataZoanthidea.

[0021] In still another embodiment of the present invention, whereinsaid composition is prepared by using whole body tissue of said species.

[0022] In still another embodiment of the present invention, whereinsaid de-odorizing agent shows no affect on the activity of said extractand fractions.

[0023] In still another embodiment of the present invention, whereinratio of said extract and/or fractions and de-odorizing agent is rangingbetween 1:1 to 1:10.

[0024] In still another embodiment of the present invention, whereinsaid composition is particularly effective against bacteria and diatoms.

[0025] In further embodiment of the present invention, a method ofproducing an extract and its fractions from Zoanthus sp, useful as ananti-fouling agent by inhibiting microbial growth.

[0026] In another embodiment of the present invention, soaking washedspecimen of said species in methanol.

[0027] In yet another embodiment of the present invention, obtainingcrude extract from the whole body tissue of said species.

[0028] In still another embodiment of the present invention, decantingthe solvent extract.

[0029] In still another embodiment of the present invention, filteringdecanted extract.

[0030] In still another embodiment of the present invention, evaporatingthe filtrate at 40° C. to obtain concentrate crude extract.

[0031] In still another embodiment of the present invention,fractionating concentrated crude extract using solvents of increasingpolarity.

[0032] In still another embodiment of the present invention, introducingsaid crude extract at concentration ranging between 0.1 to 5.0-mg/6mm-diameter disc in methanol on sterilized filter paper disc.

[0033] In still another embodiment of the present invention, evaporatingthe solvent from the disc.

[0034] In still another embodiment of the present invention, exposingmicrobes to said extract.

[0035] In still another embodiment of the present invention, laying saiddisc on agar plates seeded with test bacterium.

[0036] In still another embodiment of the present invention, running acontrol with solvent not containing extract.

[0037] In still another embodiment of the present invention, incubatingsaid plates overnight for temperature ranging between 25 to 32° C.

[0038] In still another embodiment of the present invention, measuringgrowth inhibition zone.

[0039] In still another embodiment of the present invention, determining% inhibition of microbes using said extract.

[0040] In still another embodiment of the present invention, determininganti-diatomic activity of crude extract and said fractions separately,by placing diatoms with initial cell suspension of concentration rangingbetween 100 to 5000 cells/ml in flasks.

[0041] In still another embodiment of the present invention, adding saidcrude extract of concentration ranging between 10 to 5000 ppm to saidcell suspension.

[0042] In still another embodiment of the present invention, adding saidfractions of concentration ranging between 10 to 500 ppm in separateflasks.

[0043] In still another embodiment of the present invention, incubatingflask at temperature ranging between 25 to 32° C. with 12 hrs light andnext 12 hrs dark exposure conditions.

[0044] In still another embodiment of the present invention, determining% growth inhibition after about 6 days i.e., during exponential growthphase of diatoms.

[0045] In still another embodiment of the present invention, whereinsaid species preferably used for said activity is CoelenterataZoanthidea.

[0046] In still another embodiment of the present invention, whereinsaid method is particularly effective in marine environment.

[0047] In still another embodiment of the present invention, whereinsaid method is particularly effective against bacteria and diatoms.

[0048] In still another embodiment of the present invention, whereinsoaking said washed specimen in methanol for number of days rangingbetween 5-15.

[0049] In still another embodiment of the present invention, whereinsaid species is obtained from intertidal rocks.

[0050] In still another embodiment of the present invention, whereindecanted extract is filtered through whatman filter paper I.

[0051] In still another embodiment of the present invention, whereinfiltrate is evaporated using vacuum.

[0052] In still another embodiment of the present invention, whereinsaid method uses zobell marine agar for bacterial culture.

[0053] In still another embodiment of the present invention, whereinconcentration of said extract against bacteria is preferably about 0.5mg/mm of diameter disc.

[0054] In still another embodiment of the present invention, whereinsaid extract is incubated at preferably 28° C.

[0055] In still another embodiment of the present invention, whereingrowth inhibition zone less than 1 mm not been scored,

[0056] In still another embodiment of the present invention, whereinsaid fractionating solvents are selected from a group comprisingpetroleum ether, chloroform, n-butanol, and aqueous.

[0057] In still another embodiment of the present invention, whereindissolving non-polar fraction petroleum ether in acetone (0.005-0.01ml).

[0058] In still another embodiment of the present invention, whereinbacteria are selected from a group comprising Bacillus cereus, B.circulans, pseudomonas vesicularis, and p. putida.

[0059] In still another embodiment of the present invention, whereinantibacterial activity of extract is 100%.

[0060] In still another embodiment of the present invention, whereindiatoms are selected from a group comprising Navicula subinflata andNitzschia closterium.

[0061] In still another embodiment of the present invention, whereincalculating % growth inhibition with extract and/or fractions havingmore than 50% growth inhibition is considered as active ones.

[0062] In still another embodiment of the present invention, whereinsaid extract shows 100% inhibition in diatoms at concentration of 350ppm and above.

[0063] In still another embodiment of the present invention, wherein allthe said fractions of crude extract show inhibitory activity againstdiatoms.

[0064] In still another embodiment of the present invention, whereinpetroleum ether fraction is most effective of all the fractions againstdiatoms.

[0065] In still another embodiment of the present invention, whereinpetroleum ether fraction shows 100% inhibition at concentration of about80 ppm and above against diatoms.

[0066] In still another embodiment of the present invention, whereinchloroform fraction shows about 100% inhibition in diatoms atconcentration about 200 ppm.

[0067] In still another embodiment of the present invention, whereinn-butanol fraction shows about 100% inhibition against diatoms atconcentration ranging between 150-250 ppm.

[0068] In still another embodiment of the present invention, whereinaqueous fraction shows inhibitory activity ranging between 70-85%against diatoms at concentration ranging between 140-250 ppm.

[0069] In still another embodiment of the present invention, whereinsaid extract/fractions do not have any toxic/adverse effect onnon-target organisms.

[0070] In still another embodiment of the present invention, whereinassaying microbes in a culture media using agar plate diffusion method.

[0071] In still another embodiment of the present invention, whereinsaid method is used to prevent fouling in food articles.

[0072] In still another embodiment of the present invention, whereinsaid method is used to prevent fouling in biological specimens.

[0073] In further embodiment of the present invention, the presentinvention provides a process for the preparation of antifouling crudeextract from Zoanthus sp and the said process comprising,

[0074] i) Collection of live/fresh specimens of Zoanthus sp fromintertidal rocks.

[0075] ii) Washing the specimens clean with fresh sea water and soakingin methanol for about 10 days iii) Filtering the extract through Whatmanfilter paper I and vacuum evaporating at 40^(U)C. to concentrate tocrude residue. In an embodiment, the crude extract of Zoanthus sp isexhibiting antibacterial property.

[0076] In a preferred embodiment, the crude extract of Zoanthus sp isinhibiting growth of fouling bacteria such as Bacillus cereus, Bacilluscirculans, Bacillus pumilus, Pseudomonas vesiciulans and Pseudomonasputida.

[0077] In another embodiment, the crude extract of Zoanthus sp isinhibiting growth of fouling diatoms at all concentrations.

[0078] In a preferred embodiment, the crude extract of Zoanthus sp iscompletely inhibiting growth of diatoms, Navicula subinflata at andabove 600 ppm concentration and Nitzschia closterium at and above 400ppm concentration.

[0079] In still another embodiment of the present invention, a processfor fractionating the crude antifouling extract from Zoanthus sp basedon increasing polarity of the solvents, viz. petroleum ether,chloroform, n-butanol and aqueous fractions and testing the antifoulingproperty of all these fractions against the fouling diatoms Naviculasubinflata and Nitzschia closterium, the said process comprising,obtaining crude extract from live/freshly collected Zoanthus sp, washingthem clean with sea water, soaking in methanol for about 10 days,filtering the extract through Whatman filter paper I and concentratingto crude residue under vacuum at 40° C.

[0080] In an embodiment, the crude extract of Zoanthus sp isfractionated based on increasing polarity of solvents into petroleumether, chloroform, n-butanol and aqueous fractions.

[0081] In another embodiment, all four fractions of the crude extract ofZoanthus sp are inhibiting the growth of fouling diatoms, Naviculasubinflata and Nitzschia closterium.

[0082] In yet another embodiment, the percentage inhibition in all thefraction is varying with concentration.

[0083] In still another embodiment, the petroleum ether fraction of thecrude extract of Zoanthus sp is more effective in inhibiting growth ofthe fouling diatoms Navicula subinflata and Nitzschia closterium.

[0084] In one more embodiment, the petroleum ether fraction of the crudeextract of Zoanthus sp completely inhibits growth of Navicula subinflataat and above 160 ppm and Nitzschia closterium at and above 80 ppmconcentrations.

[0085] In further embodiment of the present invention, a process for thepreparation of an antifouling crude extract from Zoanthus sp and testingthe said crude extract on fouling bacteria and diatoms.

[0086] The invention also relates to a process for fractionation of thecrude extract and testing the antifouling property of the fractions onfouling bacteria.

[0087] A process for the preparation of antifouling crude extract fromZoanthus sp and the said process comprising,

[0088] Collection of live/fresh specimens of Zoanthus sp from intertidalrocks.

[0089] Washing the specimens clean with fresh seawater and soaking inmethanol for about 10 days.

[0090] Filtering the extract through Whatman filter paper I and vacuumevaporating at 40° C. to concentrate to crude residue.

[0091] In still another embodiment of the present invention, wherein thecrude extract of Zoanthus sp is exhibiting antibacterial property.

[0092] In still another embodiment of the present invention, wherein thecrude extract of Zoanthus sp is inhibiting growth of fouling bacteriasuch as Bacillus cereus, Bacillus circulans, Bacillus pumilus,Pseudomonas vesiciularis and Pseudomonas putida.

[0093] In still another embodiment of the present invention, wherein thecrude extract of Zoanthus sp is inhibiting growth of fouling diatoms atall concentrations.

[0094] In still another embodiment of the present invention, wherein thecrude extract of Zoanthus sp is completely inhibiting growth of diatoms,Navicula subinflata at and above 600 ppm concentration and Nitzschiaclosterium at and above 400 ppm concentration.

[0095] In still another embodiment of the present invention, wherein-thecrude extract of Zoanthus sp may have potential use as an antifoulingagent/in aquatic environment.

[0096] In still another embodiment of the present invention, a processfor fractionating the crude antifouling extract from Zoanthus sp basedon increasing polarity of the solvents, viz. petroleum ether,chloroform, n-butanol and aqueous fractions and testing the antifoulingproperty of all these fractions against fouling diatoms, the saidprocess comprising, obtaining crude extract from live/freshly collectedZoanthus sp, washing them clean with sea water, soaking in methanol forabout 10 days, filtering the extract through Whatman filter paper I andconcentrating to crude residue under vacuum at 40° C.

[0097] In still another embodiment of the present invention, wherein thecrude extract of Zoanthus sp is fractionated based on increasingpolarity of solvents into petroleum ether, chloroform, n-butanol andaqueous fractions.

[0098] In still another embodiment of the present invention, wherein allthe four fractions of the crude extract of Zoanthus sp are inhibitingthe growth of fouling diatoms, Navicula subinflata and Nitzschiaclosterium.

[0099] In still another embodiment of the present invention, wherein thepercentage inhibitions in all the fraction is varying with cone

[0100] In still another embodiment of the present invention, wherein thepetroleum ether fraction of the crude extract of Zoanthus sp is moreeffective in inhibiting growth of the fouling diatoms Naviculasubinflata and Nitzschia closterium.

[0101] In still another embodiment of the present invention, wherein thepetroleum ether fraction of the crude extract of Zoanthus sp completelyinhibits growth of Navicula subinflata at and above 160 ppm andNitzschia closterium at and above 80 ppm concentrations.

[0102] In still another embodiment of the present invention, wherein allfractions of the crude extract of Zoanthus Sp, more particularly thepetroleum ether fraction may have potential use as an antifouling agentin the aquatic environment.

[0103] The invention is further explained with the help of the followingexamples and should not be construed to limit the scope of theinvention.

EXAMPLE 2

[0104] Zoanthus sp collected from Anjuna beach (Goa) was used for theinvention. The animals were carefully scrapped from the intertidal rocksduring the low tide period using a metal spatula. About 2 kg wet weightof the specimens were used for the study. The specimens were thoroughlywashed with fresh seawater and soaked in methanol for 6-10 days forcrude extract preparation. Extracts were obtained from the whole bodytissue. The solvent extract was decanted, filtered through Whatmanfilter paper I and vacuum evaporated at 40° C. to concentrate to crudeextract. About 30 gm of crude extract was thus obtained. This exampleillustrates the methodology used for preparation of the crude methanolextract from Zoanthus sp

EXAMPLE 2

[0105] Five bacterial isolates, all belonging to the fouling communitywere used for the assay. The bacterial assays were carried out by agarplate diffusion method (Amade et al., 1987, Mar. Biol, 94, 271-275)using Zobell marine agar (Himedia, Mumbai). Bacterial strains used forthe assay were Bacillus cereus B. circulans. B. pumilus, Pseudomonasvesicularis and P. putida. Concentrations of 0.5-mg/6 mm-diameter discof the extract prepared in methanol was introduced on sterilized filterpaper discs. After solvent evaporation, the discs were laid on agarplates seeded with the test bacterium Controls (in duplicate) containedonly the solvent. Four replicates of each concentration were used forthe assay and average values are presented. Plates were incubatedovernight at room temperature (<<28° C.). Growth inhibition zone wasmeasured in mm. (Berquist and Bedford, 1978, Mar. Biol, 46, 215-221).Inhibition zones measuring <1 mm were not scored. The results of thisactivity is presented in Table 1. This example illustrates themethodology used for carrying out the antibacterial assay utilizing theextract of Zoanthus sp. TABLE 1 Antibacterial screening with crudeZoanthus sp extract. Bacteria Inhibition Bacillus cereus + Bacilluscirculans + Bacillus pumilus + Pseudomonas vesicularis + Pseudomonasputida +

EXAMPLE 3

[0106] The diatoms, Navicula subinflata and Nitzschia closterium, bothbelonging to the fouling community, were maintained in enriched seawater (Gentile and Johnsor, 1974, in, Proceedings on a Workshop onMarine Bioassays, convened by Geraldine Cox, USA, 128). Actively growingcultures with an initial cell suspension of around 500-1000 cells ml”¹were used for the assay. Concentration series of exact ranging between200 and 1000 ppm.. Aliquots of each extract stock solution (1 cc=50 mg)prepared in distilled water was taken corresponding to the desiredexperimental concentration and the volume was adjusted to 50 ml in 125ml Erlenmeyer flasks. All assays were carried out in quadruplicate.Control flasks contained only the algae cells in medium. Flasks wereincubated at room temperature (approx. 28° C.) with a 12 hr light and 12hours dark exposure conditions. Growth inhibition was determined after 6days i.e., during the exponential growth phase in terms of the number ofattached algal cells using a Sedgwick rafter and is expressed as %growth inhibition in Table 2. Extracts that were able to produce 50%growth inhibition were considered as active ones. This exampleillustrates methodology used for carrying out the/antidiatomassay/utilizing the crude Zoanthus sp. TABLE 2 Percentage growthinhibition of diatoms at different concentrations of the crudeextract.of Zoanthus sp. % Inhibition +/− Diatom species Concentration SDNavicula subinflata 200  49.7 +/− .8  400  96.3 +/− 0.4 600 100.0 +/−0.0 800 100.0 +/− 0.0 1000 100.0 +/− 0.0 Nitzschia closterium 200  5.5+/− 8.3 400 100.0 +/− 0.0 600 100.0 +/− 0.0 800 100.0 +/− 0.0 1000 100.0+/− 0.0

[0107] The crude extract was then fractionated into four fractions usingsolvents of increasing polarity viz. petroleum ether, chloroform,n-butanol and aqueous. All fractions were tested for the activity asshown in Table 3 using the method mentioned above. The non-polarfractions that were insoluble in distilled water during the preparationo£. stock solution, were dissolved in acetone (0.005-0.01 ml). Controlflasks for these sets contained cells plus acetone (equal volume addedto make the stock solution) in the medium. This example illustrates themethodology used for fractionation of the crude extract and theirtesting for the anti-diatom assay. TABLE 3 Percentage growth inhibitionof diatoms at different concentrations of the four fractions of thecrude extract of Zoanthus sp. % inhibition ± SD Concentration PetroleumDiatom species (ppm) ether Chloroform n-butanol Aqueous Naviculasubinflata 40 22.8 ± 3.0  stimulation 2.8 ± 1.6 stimulation 80 30.5 +/−3.0  stimulation 6.7 +/− 1.6 11.3 +/− 4.2  120 99.7 +/− 0.1  stimulation15.0 ± 1.2  42.2 +/− 9.3  160 100.0 +/− 0.0  14.8 +/− 5.4  64.6 +/− 0.9 44.7 +/− 21.4 200 100.0 +/− 0.0  89.4 +/− 11.7 74.5 ± 1.2  69.0 ± 7.1 Nitzschia closterium 40 81.0 ± 5.2  stimulation 5.2 ± 6.8 23.3 ± 11.3 80100.0 +/− 0.0  44.1 +/− 8.9  14.8 ± 1.6  30.7 +/− 3.0  120 100.0 +/−0.0  50.4 ± 5.9  59.9 ± 7.2  48.7 ± 13.7 160 100.0 ± 0.0  77.7 +/− 14.8100.0 +/− 0.0  67.2 +/− 6.3  200 100.0 +/− 0.0  100.0 ± 0.0  100.0 ±0.0  77.8 ± 8.7 

EXAMPLE 5

[0108] This example illustrates the methodology used for the treatmentof results in the antidiatom assay using crude extract and of Zoanthussp.

[0109] The percentage growth inhibition was calculated using theformula,$\frac{\left( {A - O} \right) - \left( {B - O} \right)}{\left( {A - O} \right)} \times 100$

[0110] Where, O=inoculum cell numbers at zero hours

[0111] A=attached cell numbers in control flask after 6 days

[0112] B=attached cell numbers in experimental flask after 6 days

[0113] Advantages of the Present Invention:

[0114] 1. This is for the first time that the antifouling property ofthe crude extract of Zoanthus sp has been identified.

[0115] 2. Laboratory studies indicated that the crude extract ofZoanthus sp was effectively inhibiting growth of fouling bacteria anddiatoms which are the primary organisms to colonize any marinesubstratum.

[0116] 3. All the four fractions, viz. petroleum ether, chloroform,n-butanol and aqueous fractions separated from the crude extractexhibited antifouling property.

[0117] 4. Among the four fractions, the petroleum ether fraction wasfound to be more effective in inhibiting the growth of fouling diatoms.

[0118] 5. This being a natural product may not have any toxic/adverseeffects on non-target Organisms.

[0119] 6. This has the potential to form a natural compound inantifouling products.

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1. A composition comprising extract and/ or its fractions from Zoanthussp, and optionally de-odorizing agents wherein said composition isuseful as an anti-fouling agent against fouling microbes.
 2. Acomposition as claimed in claim 1, wherein said extract and itsfractions are prepared by using either polar or non-polar solvent.
 3. Acomposition as claimed in claim 1, wherein animal species preferablyused for said activity is Coelenterata Zoanthidea.
 4. A composition asclaimed in claim 1, wherein said composition is prepared by using wholebody tissue of said species.
 5. A composition as claimed in claim 1,wherein said de-odorizing agent shows no affect on the activity of saidextract and fractions.
 6. A composition as claimed in claim 1, whereinratio of said extract and/or fractions and de-odorizing agent is rangingbetween 1:1 to 1:10.
 7. A composition as claimed in claim 1, whereinsaid composition is particularly effective against bacteria and diatoms.8. A method of producing an extract and its fractions from Zoanthus sp,useful as an anti-fouling agent by inhibiting microbial growth, saidmethod comprising: (a) soaking washed specimen of said species inmethanol, (b) obtaining crude extract from the whole body tissue of saidspecies, (c) decanting the solvent extract, (d) filtering decantedextract, (e) evaporating the filtrate at 40° C. to obtain concentratecrude extract, (f) fractionating concentrated crude extract usingsolvents of increasing polarity, (g) introducing said crude extract atconcentration ranging between 0.1 to 5.0 mg/6 mm diameter disc inmethanol on sterilized filter paper disc, (h) evaporating the solventfrom the disc, (i) exposing microbes to said extract, (j) laying saiddisc on agar plates seeded with test bacterium, (k) running a controlwith solvent not containing extract, (l) incubating said platesovernight for temperature ranging between 25 to 32° C., (m) measuringgrowth inhibition zone, (n) determining % inhibition of microbes usingsaid extract, (o) determining anti-diatomic activity of crude extractand said fractions separately, by placing diatoms with initial cellsuspension of concentration ranging between 100 to 5000 cells/ml inflasks, (p) adding said crude extract of concentration ranging between10 to 5000 ppm to said cell suspension, (q) adding said fractions ofconcentration ranging between 10 to 500 ppm in separate flasks, (r)incubating flask at temperature ranging between 25 to 32° C. with 12 hrslight and next 12 hrs dark exposure conditions, (s) determining % growthinhibition after about 6 days i.e., during exponential growth phase ofdiatoms,
 9. A method as claimed in claim 8, wherein said speciespreferably used for said activity is Coelenterata Zoanthidea.
 10. Amethod as claimed in claim 8, wherein said method is particularlyeffective in marine environment.
 11. A method as claimed in claim 8,wherein said method is particularly effective against fouling microbes.12. A method as claimed in claim 8, wherein said method is particularlyeffective against bacteria and diatoms.
 13. A method as claimed in claim8, wherein soaking said washed specimen in methanol for number of daysranging between 5-15.
 14. A method as claimed in claim 8, wherein saidspecies is obtained from intertidal rocks.
 15. A method as claimed inclaim 8, wherein decanted extract is filtered through whatman filterpaper I.
 16. A method as claimed in claim 8, wherein filtrate isevaporated using vacuum.
 17. A method as claimed in claim 8, whereinsaid method uses zobell marine agar for bacterial culture.
 18. A methodas claimed in claim 8, wherein concentration of said extract againstbacteria is preferably about 0.5 mg/mm of diameter disc.
 19. A method asclaimed in claim 8, wherein said extract is incubated at preferably 28°C.
 20. A method as claimed in claim 8, wherein growth inhibition zoneless than 1 mm not been scored.
 21. A method as claimed in claim 8,wherein said fractionating solvents are selected from a group comprisingpetroleum ether, chloroform, n-butanol, and aqueous.
 22. A method asclaimed in claim 8, wherein dissolving non-polar fraction petroleumether in acetone (0.005-0.01 ml).
 23. A method as claimed in claim 8,wherein bacteria are selected from a group comprising Bacillus cereus,B. circulans, pseudomonas vesicularis, and p. putida.
 24. A method asclaimed in claim 8, wherein antibacterial activity of extract is 100%.25. A method as claimed in claim 8, wherein diatoms are selected from agroup comprising Navicula subinflata and Nitzschia closterium.
 26. Amethod as claimed in claim 8, wherein calculating % growth inhibitionwith extract and/or fractions having more than 50% growth inhibition isconsidered as active ones.
 27. A method as claimed in claim 8, whereinsaid extract shows 100% inhibition in diatoms at concentration of 350ppm and above.
 28. A method as claimed in claim 8, wherein all the saidfractions of crude extract show inhibitory activity against diatoms. 29.A method as claimed in claim 8, wherein petroleum ether fraction is mosteffective of all the fractions against diatoms.
 30. A method as claimedin claim 8, wherein petroleum ether fraction shows 100% inhibition atconcentration of about 80 ppm and above against diatoms.
 31. A method asclaimed in claim 8, wherein chloroform fraction shows about 100%inhibition in diatoms at concentration about 200 ppm.
 32. A method asclaimed in claim 8, wherein n-butanol fraction shows about 100%inhibition against diatoms at concentration ranging between 150-250 ppm.33. A method as claimed in claim 8, wherein aqueous fraction showsinhibitory activity ranging between 70-85 % against diatoms atconcentration ranging between 140-250ppm.
 34. A method as claimed inclaim 8, wherein said extract/fractions do not have any toxic/adverseeffect on non-target organisms.
 35. A method as claimed in claim 8,wherein assaying microbes in a culture media using agar plate diffusionmethod.
 36. A method as claimed in claim 8, wherein said method is usedto prevent fouling in food articles.
 37. A method as claimed in claim 8,wherein said method is used to prevent fouling in biological specimens.